Our long-term goal is to understand at the cellular level the mechanism by which the obligate intracellular parasite Toxoplasma gondii enters the wide range of cells that it can infect. T. gondii is an important pathogen in immunosuppressed patients, especially those with Acquired Immune Deficiency Syndrome. Host cell invasion is a key step in the pathogenesis of toxoplasmosis, for it is the ability of T. gondii to invade many different cell types that leads to the devastating disease seen in the immunosuppressed and the newborn. Toxoplasmosis of the central nervous system is especially prominent in AIDS. Penetration of non-phagocytic cells is an active process, requiring parasite energy, and facilitated by a substance produced by the parasite and secreted at the time of host invasion (Penetration Enhancing Factor or PEF). We have developed a series of monoclonal antibodies which recognize and interfere with the function of T. gondii PEF. We will use these antibodies to recover and purify PEF, and then will investigate its mode of action by immunochemical, biochemical, and functional asays. We will characterize the molecular weight and isoelectric point of the antigen, and examine the function of purified antigen by bioassays and by its effect on model membrane systems. We will investigate the enzymatic character of the antigen in biochemical assays. Monospecific polyclonal antisera will be produced by the immunization of rabbits with purified antigen and used to screen a T. gondii gene library to locate and clone the gene for PEF. We will then be able to sequence the gene, determine its copy number and investigate interstrain and interspecies differences. We also intend to continue studies on the mechanism of T. gondii motility. Immunoelectron microscopy and freeze fracture will be used to access the role of parasite and host cytoskeleton in the process of cell invasion. Fluorescence techniques will be used to study the membrane potential, the cellular and intravacuolar pH of this parasite and the role of ionized calcium in the process of motility and cell invasion. Because of similarities between T. gondii and the merozoites of Plasmodium and Cryptosporidium, understanding of the mechanism of cell invasion may suggest therapeutic interventions of a general nature. Characterization of an antigen which is secreted at the time of cell invasion may be of use in diagnostic tests designed to distinguish acute and chronic infection.